Microbial metabolite butyrate promotes induction of IL-10+IgM+ plasma cells

The microbially-derived short-chain fatty acid butyrate is a central inhibitor of inflammatory innate and adaptive immune responses. Emerging evidence suggests that butyrate induces differentiation of IL-10-producing (IL-10+) regulatory B cells. However, the underlying mechanisms of butyrate-driven modulation of B cell differentiation are not fully defined. Given the dominant role of regulatory plasma cells (PCs) as the main source of anti-inflammatory cytokines including IL-10 and the observation that butyrate also induces the differentiation of PCs, we here investigated the effect of the microbial metabolite butyrate on the induction of regulatory IL-10+ PCs and underlying mechanisms. Here we show that butyrate induces the differentiation of IL-10+IgM+ PCs. Ex vivo, butyrate, but hardly propionate, another microbially-derived short-chain fatty acid, induced the differentiation of IL-10+IgM+ CD138high PCs from isolated splenic murine B cells. In vivo, administration of butyrate via drinking water or by daily intraperitoneal injection increased the number of IL-10+IgM+ CD138high PCs in the spleens of Ovalbumin (Ova)/complete Freund’s adjuvant-immunized mice. The induction of these regulatory PCs was associated with an increase of anti-Ova IgM, but a reduction of anti-Ova class-switched pathogenic IgG2b serum antibodies. Based on the knowledge that butyrate inhibits histone deacetylases (HDACs) thereby increasing histone acetylation, we identified here that HDAC3 inhibition was sufficient to induce PC differentiation and IL-10+ expression. Furthermore, reduced mitochondrial superoxide levels following butyrate treatment and HDAC3 inhibition were necessary for PC differentiation, but not IL-10 expression. In summary, the microbial metabolite butyrate promotes the differentiation of IgM+ PCs and their expression of IL-10. HDAC3 inhibition may be involved as an underlying pathway for both PC differentiation and IL-10 expression, while reduced mitochondrial superoxide levels are crucial only for PC differentiation. The induction of regulatory IL-10+IgM+ PCs and the inhibition of class switching to antigen-specific pathogenic IgG subclasses might represent important pathways of butyrate to limit inflammation.


Introduction
Gut microbiome and the host's immune system are tightly intertwined in a complex network of reciprocal regulatory mechanisms in health and disease [1,2]. Microbially-derived metabolites are of crucial relevance in regulating immune homeostasis and protecting against allergy and autoimmunity [3][4][5]. Accordingly, gut microbial composition and microbial metabolites are frequently disturbed in autoimmune and allergic diseases [1,4,6,7].
Short-chain fatty acids (SCFAs), including butyrate (BA; C4) and propionate (PA; C3), are potent microbial metabolites with a wide array of immunomodulatory functions [3,5]. SCFAs are produced mainly from dietary fiber (undigested complex carbohydrates) by microbial fermentation and reach high concentrations of 60-140 mM in the colon, whereas lower concentrations are detected in the blood (micromolar range) [8,9]. Importantly, SCFAs accumulate in tissues of high immunological relevance, such as mesenteric lymph nodes and the spleen, reaching concentrations between 1 and 1.5 mmol � kg -1 in the case of BA [9]. Furthermore, oral administration of SCFAs is an effective method for increasing SCFA concentrations in these organs [9]. Thus, immunological effects of SCFAs are more likely to occur either in the gastrointestinal tract or in immunological effector organs where high concentrations of SCFAs are present rather than in the systemic circulation.
Considering the anti-inflammatory effects of SCFAs on a wide array of immune cells, modulation of the B cell compartment by SCFAs has not been fully defined [1,28]. B cells, in part due to the production of pathogenic IgG (auto-) or IgE antibodies and activation of other immune cells, are central to the pathogenesis of autoimmunity and allergy, respectively [29]. Moreover, B cell subsets, including CD138 high plasma cells (PCs), are increasingly recognized to exert anti-inflammatory functions by producing anti-inflammatory cytokines (IL-10, TGFβ, IL-35) [30][31][32] and highly glycosylated (esp. sialylated) IgG antibodies [33][34][35][36][37][38]. Thus, modulating the effector B cell phenotype towards a more anti-inflammatory state represents an intriguing target for the treatment of autoimmune and allergic diseases. Notably, BA protects against autoimmunity by increasing IL-10 + Bregs [25,27] and by supporting antibody responses both in the intestine and systemically [9,39]. However, whether induction of regulatory properties of B cells and induction of PC differentiation by BA are functionally and mechanistically related and which cellular pathways are substantially involved remains to be defined.
Here we investigated the effects of BA on PC differentiation and the expression of antiinflammatory cytokines, in particular IL-10. We show that BA increases the differentiation of IL-10 + IgM + PCs both ex vivo and in vivo which was associated with a reduction of antigen-specific IgG2b class switching. Mechanistically, BA led to increased histone acetylation in B cells, which is a downstream effect of HDAC inhibition, and inhibition of HDAC3 was identified to be sufficient for the induction of PCs and IL-10 expression underlining the importance of epigenetic modulation of B cell function. Reduced mitochondrial superoxide levels after BA treatment and following HDAC3 inhibition were necessary for PC differentiation, but not for IL-10 expression, indicating that these processes are initiated in parallel and not in consequence of one another.

Mice
C57BL/6 wt mice (WT) were purchased from Charles River Laboratories (Bar Harbor, ME, USA). IL-10 GFP reporter (Vert-X) mice were originally provided by Prof. Christopher L. Karp (Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA) and bred inhouse [40]. Mice were kept at the local animal facilities of either the University of Luebeck or Cincinnati Children's Hospital Medical Center (Cincinnati, OH, USA) under specific-pathogen-free conditions at 22˚C with a 12h light/dark cycle and with food and water ad libitum. Mice were kept and all experiments were performed in strict accordance with governmental and institutional guidelines with approval by the governmental administration and the animal research ethics boards of the corresponding ministries of the state of Schleswig-Holstein, Germany (approval number: 51-5/2019) or the Cincinnati Children's Hospital Medical Center's Institutional Animal Care and Use Committee (IACUC 2020-0034). Monitoring of the health and well-being of the mice was conducted at least daily by a qualified person. 8-to 12-weekold mice were used for in vivo experiments. 8-to 16-week-old mice were used for ex vivo experiments. All efforts were made to minimize and alleviate distress and suffering. Refined aspects of study planning, housing, and husbandry were applied. Power analysis was conducted prior to the study to reduce the number of animals to the necessary minimum for reliable results. No harmful noise, vibrations, or lighting were present during the study. Habituation and husbandry handling was combined with handling for research purposes wherever possible. Mice were anesthetized and analgized by injection of ketamine hydrochloride (80 mg/kg body weight; Medistar, Germany) and xylazine hydrochloride (10 mg/kg body weight; Bayer, Germany). Mice were sacrificed according to the respective governmental and institutional guidelines either by cervical dislocation or CO 2 exposure. B cell isolation using magnetic activated cell sorting B cells were isolated from murine spleens by depleting non-B cells using magnetic activated cell sorting (Mouse B cell isolation kit, #130-090-862, Miltenyi Biotec, Germany) following the manufacturer's protocol. In short, splenic cells were obtained by passing the organ twice through 70 μm nylon filters (352350, Fisher Scientific, Germany). Non-B cells were labeled with biotinylated antibodies directed against several surface antigens that are not expressed by murine B cells (CD43, CD4, and Ter-119). Magnetic beads conjugated with anti-biotin antibodies were added as a secondary labeling agent. The cell suspension was then added to separation columns (LS columns, #130-042-401, Miltenyi Biotec, Germany) in a strong magnetic field (QuadroMACS Separator, #130-090-862, Miltenyi Biotec, Germany) capturing cells labeled with biotinylated antibodies and bound to magnetic anti-biotin beads. The non-labeled B cells were collected with the flow-through achieving a B cell purity of min. 95%.

Cell culture
Isolated B cells were cultured in RPMI medium with 10% FCS, 100 μM 2-mercaptoethanol, 1 mM HEPES, and 100 U/ml Penicillin/Streptomycin (later referred to as B cell medium). B cells were cultured at 10 5 to 10 6 cells per ml. For basic PC-inducing conditions in all cell culture experiments, the medium was supplemented with 3 μg/ml LPS plus 10 ng/ml IL-6 (#216-16, Peprotech, USA) plus 100 nM all-trans Retinoic Acid (#R2625, Sigma-Aldrich, USA). Sodium butyrate (BA) and sodium propionate (PA) working solutions were freshly prepared and added directly to cell culture on day 0 of the experiments to final concentrations of 0.1 mM to 0.5 mM. 2-DG (200 μM), PMA (100 ng/ml), RGFP966 (10 μM), TSA (90 nM), and GPR43/ FFAR2 agonist (1 μM) working solutions were freshly prepared and added directly to cell culture on day 0 of the experiments to the indicated final concentrations. Cells were cultured at 37˚C and 5% CO 2 (Heracell VIOS 160i, Thermo Fisher Scientific, USA) for up to four days as described for each experiment.
Immunization and BA treatment in vivo 8 to 12-week-old WT or IL-10-reporter mice were treated with BA and immunized i.p. with 100 μg Ova/CFA. In brief, BA or the respective control was administered to the mice continuously starting 7 days before immunization in two different routes of administration: i) BA was dissolved in designated drinking water (DW) from the animal facility at 150 mM and provided ad libitum, DW was used as the control; ii) BA was administered by daily intraperitoneal (i.p.) injection (100 mg/kg body weight); PBS was injected as the vehicle control. Mice were weighed every 2-3 days and in the case of DW experiments, the consumption of water was recorded. 12 days after Ova/CFA immunization all mice were sacrificed, and spleens and sera were sampled for further analysis.

Flow cytometry
Single-cell suspensions from cell culture were prepared by washing the cells twice (5 min, 400 x g, 4˚C) and resuspending them in FACS buffer. Single-cell suspensions from murine spleens were prepared by passing the organ through a 70 μm nylon filter (#352350, Thermo Fisher Scientific, Germany), followed by incubation in ammonium-chloride-potassium lysing buffer (#A1049201, Thermo Fisher Scientific, USA) for 5 min at room temperature to remove erythrocytes. Afterward, cells were washed in FACS-buffer and passed again through a 70 μm nylon filter to remove any remaining tissue debris or cellular aggregates. The numbers of viable cells were determined and normalized to 0.2 to 1 x 10 6 cells per sample for cell culture experiments before cell staining commenced.

qRT-PCR
Cultured B cells were lysed and RNA was extracted using the innuPREP RNA Mini Kit 2.0 (Analytik Jena, Germany) according to the manufacturer's instructions and treated with amplification grade DNase I (Thermo Fisher Scientific, USA). RNA contents were quantified using a Nanodrop 2000 (Thermo Fisher Scientific, MA, USA) and stored at -80˚C until further usage. 1 μg of isolated RNA per sample was added to RNase/DNase-free reaction tubes, and RNase/DNase-free water was added up to a volume of 11.5 μl. Next, 8.5 μl of freshly prepared Master Mix were added to each sample, containing 0.5 μl RNase inhibitor (Ribolock, 40 U/μl, #EO0384, Thermo Fisher, USA), 2 μl dNTP mix (dNTP set, #20-2011, 100 mM each, peq-GOLD, VWR International, USA), 1 μl reverse transcriptase (RevertAid H Minus Reverse Transcriptase, #EP0442, 200 U/μL, Thermo Fisher, USA), 4 μl 5x Reaction Buffer (included in RevertAid H Minus Reverse Transcriptase Kit, #EP0442, Thermo Fisher, USA), and 1 μl Oligo (dT) (biomers.net, Germany). The reaction mix was incubated for 60 min at 42˚C for reverse transcription, 10 min at 70˚C for enzyme inactivation, diluted 1:5 in DNase-free water, and stored at -20˚C until further usage. 2 μl cDNA sample per technical replicate were added to a 96-well PCR plate (Sarstedt, Germany). 10 μl PerfeCTa SYBR Green SuperMix (#733-1251, VWR International, USA) as well as 0.25 μl forward and reverse primers (final concentration of 125 nM each) were added following manufacturer's instructions. 7.5 μl DNAse-free water was added for a final reaction volume of 20 μl per well. The primer pairs were designed using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), purchased from Metabion (Germany) and are shown in Table 1. The StepOnePlus Real-Time PCR System and Software (Thermo Fisher Scientific, USA) were used to conduct the qPCR following the manufacturer's recommendations. At least two technical replicates were included for each sample. mRNA expression of each gene was compared to β-actin expression (endogenous housekeeping gene control) using the ddCT method and calculated as fold change of control.

ELISA
ELISAs were performed following previously published methods [41] and slightly modified where necessary. In brief, blocking, serum dilutions, and the addition of detection antibodies were conducted in 2.5% milk powder in PBS. For anti-Ova antibody measurements, 10 mg/mL (for IgM, IgG1, IgG2b, and IgG2c) or 40 mg/mL (for IgA) of Ova were used to generate Ovacoated ELISA-plates. For the total IgM antibody measurements, an anti-murine IgM antibody from Bethyl Laboratories (Montgomery, USA) was used in a concentration of 5 μg/ml. After incubation with the indicated serum dilutions (1:100 for IgA, IgM, IgG2b, IgG2c; 1:1000 for IgG1) bound antibodies were detected with horseradish peroxidase (HRP)-coupled polyclonal goat anti-mouse IgA-, IgM-, IgG1-, IgG2b-, or IgG2c (the IgG2a haplotype in C57BL/6 mice)specific antibodies purchased from Bethyl Laboratories (Montgomery, USA). The detection antibodies were diluted according to the manufacturer's instructions. After incubation with the 3,3 ,5,5´-tetramethylbenzidine (TMB) substrate (BD Biosciences, San Diego, USA), the reaction was stopped using the same volume of 4,2% H 2 S0 4 . Eventually, the OD was measured at 450 nm. Table 1. qRT-PCR primers. Gene targets, forward and reverse RT-PCR primer sequences, and product lengths.

Seahorse mitochondrial stress assay
To analyze mitochondrial respiration of B cells, oxygen consumption rates (OCR) were measured using the Seahorse Mito Stress kit (Agilent, USA). All experiments were performed following an optimized protocol for analyzing lymphocytes previously published elsewhere [42]. B cells were isolated from murine spleens using magnetic activated cell sorting and allowed to rest in B cell medium (as described above) for one hour. Negative magnetic activated cell sorting was used because

Statistical analysis
Sample sizes were determined based on ex vivo data and effect sizes from preliminary experiments. The statistical tests employed were selected depending on the number of groups being compared and data distribution. For normally distributed data, either paired or unpaired Student's t-test was used as applicable. For comparisons of more than two groups, either one-way ANOVA or a mixed-effects model for paired comparisons were employed with Geisser-Greenhouse correction. Tukey's post-hoc test was applied to follow up on significant results for pairwise comparisons of interest. For non-parametric data sets, the Mann-Whitney U was employed. Statistical analysis was performed using Prism 9.0.0 (GraphPad Software, Inc.). For each graph, data sets from 1-3 representative experiments are shown. If not stated otherwise, experiments were at least replicated twice and are presented as the mean ±SEM. � p < 0.05, �� p < 0.01, and ��� p < 0.001.

BA induces CD138 high PCs and the expression of Prdm1 and Irf4 ex vivo
SCFAs increase systemic antibody production in mice invoking potential impact on PC frequencies [9,39]. To test whether PA and/or BA directly induce differentiation of B cells towards PCs, murine B cells were cultured ex vivo in PC-inducing conditions and treated with increasing concentrations of PA or BA for 4 days. The administration of 0.1 or 0.25 mM PA did not change the frequency of induced PCs (defined by CD138 high expression). Only 0.5 mM PA showed a slight increase in the frequency of CD138 high PCs (p = 0.0215, Fig 1A and 1B).
In contrast, BA showed a strong dose-dependent effect on the induction of CD138 high PCs (Fig 1A and 1B). At a concentration of 0.5 mM, BA was sufficient to double the frequency of CD138 high PCs (p < 0.0001). The addition of 0.5 mM PA to 0.5 mM of BA did not induce an additional effect on PC differentiation (Fig 1A and 1B). Taken together, while PA shows only a slight effect, BA effectively increases CD138 high PC differentiation from naïve B cells. PC differentiation is regulated by several transcription factors including Interferon regulatory factor (Irf) 4, Irf8, B-cell lymphoma 6 protein (Bcl6), X-box binding protein 1 (Xbp1), and PR domain zinc finger protein 1 (Prdm1) [43]. Given the ability of BA to increase frequencies of CD138 high PCs, gene expression of transcriptional regulators of PC differentiation was examined in splenic B cells treated with 0.5 mM BA under PC-inducing conditions for one day without additional cell sorting after differentiation. Notably, the expression of Prdm1, a master transcriptional regulator of PC differentiation, was upregulated 2.5-fold after incubation with BA (p = 0.0359; Fig 1C). Additionally, Irf4, an essential inducer of PC differentiation [44], was increased approximately 5-fold (p = 0.0220; Fig 1C) and Irf8, a potent repressor of PC differentiation and inductor of B cell anergy and non-PC lineages [45], was not detectable in any sample after BA treatment (Fig 1C). No changes were found in the gene expression of Bcl6 and Xbp1 (Fig 1C), which are closely linked to inhibition [46] and induction of PC differentiation [47], respectively. Interestingly, gene expression of Aicda, which is crucially involved in antibody class switch recombination in the germinal center [48], was significantly reduced in BA-treated  Fig 1C). Thus, BA treatment modifies B cell gene expression profiles of important transcriptional regulators towards PC differentiation including Prdm1 and Irf4, while repressing the negative regulator Irf8 and the class switch inducer Aicda.

BA but not PA induces IL-10 expression in B cells and PCs ex vivo
PCs can induce anti-inflammatory effects by the production of anti-inflammatory cytokines including IL-10, TGFβ, and IL-35 (EBI3/p35). Thus, we next analyzed cytokine gene expression in B cells after four days of treatment with PA and/or BA by qPCR. Treatment with increasing concentrations of PA did not induce Il10, Tgfβ1, Ebi3, or p35 gene expression (Fig  2A). Conversely, treatment of B cells with BA significantly increased gene expression of Il10 (0.5 mM BA: p = 0.0003), Tgfβ1 (0.5 mM BA: p = 0.0004), and Ebi3 (0.5 mM BA: p = 0.0003) in a dose-dependent manner (Fig 2B).
To evaluate the effect of BA treatment on IL-10 expression at the protein level, isolated splenic B cells from IL-10-reporter mice were stimulated, and IL-10 expression was evaluated by flow cytometry. After four days of culture, PA treatment did neither influence IL-10 reporter gene expression in all B cells nor CD138 high PCs (Fig 2C-2F). In contrast, BA treatment increased the frequency of IL-10 + cells among all B cells as well as CD138 high PCs in a dose-dependent manner (0.5 mM BA: p = 0.0097 for IL-10 + cells of all CD138 high PCs and p = 0.0016 for IL-10 + CD138 high PCs of all B cells, Fig 2C-2F). When 0.5 mM PA was added to 0.5 mM BA, IL-10 expression in CD138 high PCs, as well as the frequency of IL-10 + CD138 high PCs of all B cells, was only modestly increased compared to 0.5 mM BA (Fig 2D and 2F).
To further characterize the IL-10 + CD138 high PCs present in our B cell culture, the surface expression of IgM was compared between IL-10and IL-10 + CD138 high PCs. Strikingly, IL-10 + CD138 high PCs showed significantly higher expression of IgM on their surface (p <0.0001; Fig  2G) indicating that the IL-10 + PCs were mainly IgM + PCs. Together these ex vivo data suggest that in particular BA, but hardly PA, induce PC differentiation and their expression of antiinflammatory cytokines including IL-10 at the transcriptional and protein level, indicating the potential of BA to induce regulatory IL-10 + IgM + PCs.

BA induces IL-10 + CD138 high PCs in vivo
Since BA induced CD138 high and IL-10 + CD138 high PCs ex vivo, the effects of BA on PC differentiation and IL-10 expression were investigated in vivo. BA was administered to mice either enterally via supplementation of drinking water (DW) with 150 mM of BA or systemically via daily intraperitoneal (i.p.) injection (100 mg/kg body weight). No significant changes in body weight or drinking volume were observed during BA treatment (S1 Fig). After seven days of BA treatment, mice were immunized with Ovalbumin (Ova)/CFA to induce a systemic immune reaction with increased PC differentiation. We evaluated the frequencies of CD138 high PCs and IL-10 + CD138 high PCs in the spleen of immunized mice treated with BA or vehicle control 12 days post-immunization.
Enteral delivery of BA significantly increased the frequencies (p = 0.0005) and absolute numbers (p = 0.0478) of splenic CD138 high PCs compared to control animals (Fig 3A and 3B). Similarly, splenic CD138 high PC frequencies (p = 0.0461) and absolute numbers (p = 0.0014) were elevated after daily BA i.p. injection compared to the PBS vehicle control (Fig 3C), indicating increased differentiation of PCs regardless of the route of administration. Furthermore, enteral BA administration increased the proportion (p = 0.0322) and absolute cell numbers (p = 0.0138) of IL-10 + CD138 high splenic PCs (Fig 3D and 3E). In addition, i.p. administration of BA increased IL-10 + CD138 high splenic PC numbers (p = 0.0014), whereas the frequency of IL-10 + CD138 high PCs was only modestly increased in this setting compared to PBS-treated controls (Fig 3F). These data demonstrate the ability of BA to induce PCs and their IL-10 expression in vivo.

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injection: 44.5%) in BA-treated mice (p < 0.0001 for both treatments; Fig 4B-4D left panels). These differences were reflected also by the median fluorescence intensities (MFI) of IgM in IL-10and IL-10 + CD138 high PCs (p < 0.0001 for both treatments; Fig 4C, 4D right panels). Similarly, the frequency of IL-10 + PCs, as well as the IL-10 GFP MFI, were significantly higher among IgM + CD138 high PCs compared to IgM -CD138 high PCs in BA-treated mice (p < 0.0001 for both treatments; Fig 4E-4G). Furthermore, administration of BA increased the frequency of IL-10 + IgM + CD138 high PCs (DW: p = 0.0290; i.p.: p = 0.0117) and IL-10 GFP MFI in IgM + CD138 high PCs in comparison to control treatment regardless of the route of administration (DW: p = 0.0093; i.p.: p = 0.0497; Fig 4H and 4I left and center panels). Accordingly, treatment with BA via drinking water induced a significant increase of total serum IgM levels (p = 0.0057, Fig 4H right  panel), while i.p. injection of BA slightly, but not significantly increased serum IgM levels (p = 0.1796, Fig 4I right panel). These data indicate that BA-induced IL-10 + CD138 high PCs preferentially express IgM in vivo.

BA induces anti-Ova IgM but inhibits class switching to pathogenic anti-Ova IgG2b upon Ova/CFA immunization
Next, we analyzed the influence of BA on the Ova-specific B cell response upon Ova/CFA immunization. To identify Ova-specific CD138 high PCs 12 days after Ova/CFA immunization, cells were additionally stained extra-and intracellularly with fluorophore-conjugated Ova. Enteral administration of BA increased the frequency of Ova-specific PCs among all CD138 high PCs (p = 0.0139), as well as the absolute number of Ova-specific CD138 high PCs (p = 0.0389, Fig 5A and 5B), while i.p. administration of BA did not increase the frequency, but the absolute number of Ova-specific PCs (p = 0.0436, Fig 5C). Serum levels of anti-Ova IgA (Fig 5D) and anti-Ova IgG1 levels (Fig 5F) remained unchanged after the application of 150 mM BA in drinking water. However, anti-Ova IgM levels were significantly elevated (p = 0.0069, Fig 5E). Moreover, anti-Ova IgG2b (p = 0.0270, Fig 5G) and in tendency anti-Ova IgG2c (Fig 5H) levels were reduced after administration of 150 mM BA in drinking water. These differences were also found in tendency, but without statistical significance after i.p. injection of BA (Fig 5I-5M). Thus, the induction of IL-10 + IgM + CD138 + PCs after BA treatment was associated with increased systemic anti-Ova IgM levels and inhibition of class switching of antigen-specific antibodies to the pathogenic IgG2b subclass particularly after enteral application of BA.

Specific inhibition of HDAC3 is sufficient to induce IL-10 + PCs
Next, we investigated the effector mechanisms of BA. GPR43/FFAR2 activation is an underlying mechanism for the immunomodulatory effects of BA on T cells and neutrophils [3]. In contrast to other SCFA-receptors (Gpr41, Gpr109a, or OLFR78), GPR43 is expressed also on activated murine B cells [9]. Thus, we next investigated whether GPR43 activation might be involved in BA-mediated PC induction. Treatment of isolated murine B cells with 0.  49]). These data suggest that GPR43 activation is not sufficient to induce PC differentiation similar to BA in murine B cells.
As immunomodulatory functions of BA are also linked to the inhibition of class I and II HDACs [3,50], the effects of BA on histone acetylation in isolated murine B cells were analyzed using a fluorophore-conjugated antibody against the acetylated lysine residue at N-terminal position 27 of the histone protein H3 (H3K27ac). Increasing concentrations of PA did not affect H3K27 acetylation, while 0.5 mM BA significantly increased the frequency of H3K27ac + -stained B cells from 4% to 22% (p = 0.0274) similar to the effects of HDAC3 inhibitor RGFP966 (Fig 6A and 6B), indicating HDAC-inhibitory properties of BA on murine B cells.
Considering the HDAC-inhibitory capacity of BA, we examined next whether HDAC inhibition recapitulates the induction of IL-10 + PCs by BA. Employing HDAC-specific inhibitors we examined whether HDAC inhibition is sufficient to induce regulatory PCs under the same conditions as BA ex vivo. Indeed, incubation of isolated splenic B cells with Trichostatin A (TSA), a chemical inhibitor of class I and II HDACs, was sufficient to recapitulate the induction of CD138 high PCs by BA (p = 0.0007; Fig 6C). Among all HDAC isotypes, HDAC3 inhibition is suggested to be specifically responsible for immunomodulatory effects of BA, but not PA [51,52]. Therefore, to examine the sufficiency of HDAC3 inhibition on inducing PC differentiation isolated murine B cells were treated with the specific HDAC3-inhibitor RGFP966. Notably, RGFP966 treatment was sufficient to induce CD138 high PCs (Fig 6C). Together, these data indicate that HDAC3 inhibition plays an important role in the induction of CD138 high PCs.
To examine whether HDAC3 inhibition was furthermore sufficient to recapitulate the induction of the anti-inflammatory cytokines Il10, Tgfβ1, and Il35 by BA, gene expression

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levels after treatment of B cells with RGFP966 for four days were determined by qPCR. Indeed, while p35 expression was not changed significantly, the expression levels of Il10 (p = 0.0284), Tgfb1 (p = 0.0055), and Ebi3 (p = 0.0087) were increased significantly (Fig 6D). Moreover,

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RGFP966 treatment increased the frequencies of IL-10 + cells among all PCs (p = 0.0007) and IL-10 + CD138 high PCs among all B cells (p < 0.0001) as shown with isolated B cells from IL-10-reporter mice (Fig 6E and 6F) recapitulating the effects observed from BA treatment. Combined, these results indicate that HDAC3 inhibition but not GPR43 activation is sufficient to induce CD138 high PCs, the expression of anti-inflammatory cytokines, and the induction of IL-10 + PCs in isolated B cells similar to the effects observed for BA.

BA decreases mitochondrial metabolism and respiration in B cells
BA modulates mitochondrial metabolism in various tissues including skeletal muscle [53], brown adipose tissue [53], liver [54], and colon [55]. Mitochondrial function is central in regulating PC differentiation [56]. Thus, we next examined the impact of BA treatment on B cell mitochondrial metabolism by conducting mitochondrial stress assays. Isolated splenic B cells were subjected to BA treatment for one day (0.5 mM) and mitochondrial respiration was measured using Seahorse Extracellular Flux Analysis (Agilent, USA) (Fig 7A). In short, extracellular flux analysis allows for the measurement of oxygen consumption rate (OCR) as a surrogate of mitochondrial metabolism. During the assay, the mitochondrial chain is modified to enable the measurement of several parameters of mitochondrial respiration, including ATP production, proton leak, spare respiratory capacity, as well as baseline and maximal respiration.
At baseline, the OCR representing basal respiration in isolated B cells decreased from 113.4 to 63.3 pmol/min after treatment with 0.5 mM BA (p = 0.0170; Fig 7B). Accordingly, ATPproduction (p = 0.0105; Fig 7C) was significantly reduced. Similarly, maximal respiration after uncoupling of the respiratory chain by FCCP (p = 0.0235; Fig 7D) was decreased, whereas no significant differences were found for spare respiratory capacity, coupling efficiency, and proton leak (Fig 7E-7G). Together, these data suggest that mitochondrial metabolism of isolated B cells, which is deeply involved in deciding B cell fate [56], is significantly inhibited after 1 day of incubation with 0.5 mM BA.

BA and specific HDAC3 inhibition decrease mitochondrial superoxide levels to induce PCs
Mitochondrial respiration is tightly linked to the production of reactive oxygen species (ROS), especially superoxide. Notably, low mitochondrial respiration predetermines B cell differentiation towards PCs [56]. Thus, we next analyzed the effects of BA on mitochondrial activity and superoxide production by flow cytometry. A combination of fluorescent stains specific for mitochondrial mass (MT Green) and mitochondrial membrane potential (TMRE) was used to evaluate mitochondrial activity. One day of BA treatment decreased the proportion of the P1 population representing MT high TMRE high B cells with highly active mitochondria from 40.9 to 35.4% (p = 0.0497, Fig 8A and 8B), while the P2 population representing MT low TMRE low B cells with less mitochondrial activity increased from 20.1 to 26.5% (p = 0.0232, Fig 8A and  8C). The MFI ratio of TMRE to MT green, which is a parameter used to determine the activity of mitochondria in relation to total mitochondrial mass, decreased with BA treatment (p = 0.0147, Fig 8D). Together these data indicate that a decrease in active mitochondrial mass correlates with the mitochondrial respiratory capacity following BA treatment (Fig 7A-7G).
Next, specific staining for mitochondrial superoxide (MitoSOX) was used as a surrogate for mitochondrial superoxide levels given their inhibitory influence on PC differentiation [56]. BA treatment reduced MitoSOX MFI by 25% (p < 0.0001, Fig 8E). Furthermore, this effect was recapitulated by a 42% decrease of MFI after specific inhibition of HDAC3 by RGFP966 (p = 0.0110, Fig 8F), indicating a reduction of mitochondrial superoxide levels by BA.
To investigate whether the reduction in superoxide levels is merely a byproduct or indeed necessary for BA effects on PC and IL-10 induction, PMA, a well-established inducer of mitochondrial superoxide [57][58][59], was used. PMA alone did not significantly change the proportion of CD138 high PCs compared to the control. However, PMA completely abrogated BAinduced PC differentiation (BA vs. BA+PMA: p = 0.0004; Fig 8G and 8H), whereas BAinduced IL-10 expression was not significantly affected by PMA (Fig 8G and 8I). Similar results were also obtained when 2DG was used as an inducer of mitochondrial ROS [56,60,61] and RGFP966 for inhibition of HDAC3 (S3 Fig). In summary, BA reduction of mitochondrial activity and superoxide production may act as a key factor in determining B cell differentiation towards PCs. Accordingly, abolished induction of PCs by BA after treatment with PMA or 2DG indicates that reduced superoxide levels are likely necessary for BA-induced PC differentiation, but not for the induction of IL-10.

Discussion
Here, we show that BA enhances the differentiation of IgM + CD138 high PCs, IL-10 expression, and IL-10 + IgM + CD138 high PCs. Ex vivo studies indicated that specific HDAC3 inhibition was sufficient to elicit both effects similar to BA, whereas reduced mitochondrial superoxide levels following HDAC3 inhibition were necessary for PC differentiation, but not IL-10 expression. The crucial relevance of HDAC3 for PC differentiation is underlined by only modest effects when PA was used. Although PA is a similarly potent HDAC inhibitor, it specifically does not inhibit the function of HDAC3 [51]. The application of a specific inhibitor of HDAC3, or BA, to induce regulatory IL-10 + IgM + PCs might therefore represent an interesting opportunity for novel treatment options and could be investigated in models of B cell-dependent inflammatory autoimmune diseases [29], e.g. models of rheumatoid arthritis [62], systemic lupus erythematosus [62], or multiple sclerosis [63].
In isolated murine B cells and Ova/CFA-immunized mice, BA promoted the differentiation of CD138 high PCs. Notably, enteral or systemic administration of BA only 7 days before Ova/ CFA immunization efficiently induced the observed effects, indicating a short-term BA application to be sufficient to induce changes in PC differentiation. Supporting evidence stems from a previous study demonstrating that SCFA administration via a high-fiber diet or drinking water supports host IgA and IgG antibody responses, also demonstrating increased PC differentiation [39]. Inhibition of HDACs by SCFAs may provide an underlying mechanism in their studies [39]. These results were further corroborated by Sanchez et al. showing increased PC differentiation in low doses of SCFAs in drinking water [9]. However, opposing effects with reduced PC differentiation were observed when a highly concentrated mixture of SCFAs and butyrate prodrugs (140mM BA and 150mM PA, 20 mg/ml tributyrin emulsion) was administered [9]. Taking into account that the different SCFAs might/can induce distinct mechanisms, mixtures of different SCFAs might result in different effects possibly explaining discrepant observations in such studies. Furthermore, recent studies with dietary fiber have shown that certain gut bacteria can generate BA, whereas other gut bacteria primarily generate the SCFAs PA or acetate (C2) [3][4][5]. Different compositions of the microbiome might therefore generate distinct SCFA proportions with distinct influences. Shifting the gut microbiome to BA-generating species might therefore be an attractive approach to induce regulatory IL-10 + PCs for the treatment of inflammatory immune diseases.
Since regulatory CD138 high PCs have been identified as the major source of B cell-derived IL-10 and IL-35 [64][65][66][67] we analyzed the effects of BA on the expression of anti-inflammatory cytokines in PCs. BA increased the expression of such cytokines (e.g., IL-10) ex and in vivo. Supporting data stems from a recent, well-executed study by Luu et al., reporting that BA, as well as the less studied SCFA pentanoate (C5), were able to effectively induce the expression of IL-10 in B cells and that pentanoate-treated B cells were able to suppress autoimmunity in a murine model of multiple sclerosis [25]. In this publication, however, the possible expression of additional Breg and PC markers and the secretion of antibodies have not been investigated, allowing for speculation, whether the induced cells might in fact be regulatory IL-10 + PCs [25]. Furthermore, BA induced IL-10 + Bregs in a model of Sjögren's Syndrome effectively ameliorating the disease, highlighting the potential therapeutic implications of inducing Bregs in autoimmune diseases [27].
Further characterization of BA-induced PCs revealed that approximately 90% of the splenic IL-10 + PCs expressed IgM in vivo. Similarly, IL-10 expression was observed in significantly higher frequencies in IgM + PCs. These results are corroborated by previous literature reporting Breg subsets to frequently express IgM [68]. Most notably, anti-inflammatory IL-10 and IL-35-producing PCs, which are the main source of these cytokines in models of infection and autoimmunity, expressed IgM at exceptionally high levels [65]. The number of splenic IL-10 + IgM + PCs and the expression of IL-10 in splenic IgM + PCs as determined by fluorescence intensity of IL-10 GFP were significantly increased after the administration of BA by drinking water or i.p. injection, underlining the capacity of BA to induce IgM + PCs of a regulatory phenotype. Accordingly, BA increased the total serum IgM levels in our studies.
By further investigating the anti-inflammatory effects of BA, we identified that the BAinduced IL-10 + IgM + PCs were associated with the generation of more Ova-specific PCs and anti-Ova IgM serum levels in Ova/CFA-immunized mice. Furthermore, class switching to anti-Ova IgG2b and in tendency IgG2c was decreased after BA treatment via drinking water, matching reduced Aicda expression in vitro. Notably, murine IgG2b and IgG2c are the two IgG subclasses that are particularly associated with proinflammatory properties, such as activation of the complement system [37], and various immune cells via high affinity-binding to activating Fcγ receptors [36,37,69,70]. BA-induced inhibition of switching to antigen-specific IgG subclasses might be an IL-10-dependent second pathway of BA to inhibit inflammatory immune responses.
The role of BA as an inhibitor of HDACs in various cell types including B cells is well established [25,71]. Here, we demonstrated increased histone (H3K27) acetylation as a surrogate for HDAC inhibition. Similarly, HDAC3 knockout in murine T cells leads to increased H3K27 acetylation and, notably, Prdm1 transcription [72]. Additionally, HDAC3 inhibition has been shown to be the culprit of the immunomodulatory effect of BA on macrophages [52], while H3K27 acetylation is associated with CD138 expression during PC differentiation [73]. Accordingly, the unspecific HDAC inhibitor TSA and the HDAC3 inhibitor RGFP966 recapitulated the effects of BA on regulatory PC differentiation from isolated murine B cells ex vivo. Gene expression of master regulators of PC differentiation Prdm1 and IRF4 were increased in isolated murine B cells after one day of culture with BA, while expression of the negative regulator IRF8 was not detectable. In contrast to BA, PA was not an effective inducer of IL-10 + PC differentiation or H3K27 acetylation and is ineffective as an HDAC3 inhibitor as well [51,52] further supporting a central role of HDAC3 inhibition for the observed effects.
Mitochondrial ROS production plays a significant role in the regulation of stem cell differentiation [74] and reduced ROS levels favor PC differentiation from activated B cells by increasing Prdm1 expression [56]. BA-inhibited mitochondrial metabolism and superoxide production in B cells likely contribute to the increased PC differentiation observed here. Similarly, HDAC3 inhibition by RGFP966 reduced mitochondrial superoxide levels. Accordingly, ROS production has been recently shown to decrease in the absence of HDAC3 in macrophages [75] linking HDAC3 inhibition by BA and reduced ROS levels. The abrogation of BAinduced PC differentiation by the mitochondrial superoxide inducers PMA [57][58][59] and 2DG [56] further indicates that decreased mitochondrial superoxide levels are necessary for BAinduced PC differentiation. However, the proposed underlying mechanisms are based mainly on ex vivo data and need verification in additional in vivo studies. Interestingly, PMA and 2DG did not abrogate the expression of IL-10 in B cells similarly to PC differentiation. Thus, reduced mitochondrial superoxide levels seem to be necessary for the induction of PCs by BA, whereas IL-10 expression is likely induced independently from mitochondrial superoxide levels. It might therefore be possible that BA-induced reduction of mitochondrial superoxide levels induces regular PCs and that another unknown anti-HDAC3-dependent mechanism induced by BA elicits the anti-inflammatory phenotype including IL-10 expression.
In this study, BA induced the differentiation of IL-10 + IgM + PCs in short-term experiments in adult mice, most likely due to HDAC-related epigenetic mechanisms (i.e., increased histone acetylation). Previous literature has suggested that epigenetic changes induced by microbiallyderived BA in early life lead to long-term protection against allergic diseases [76][77][78][79]. These effects have mainly been attributed to the correction of the exacerbated Th2-response observed in allergy [79,80]. However, regulatory B cells play a crucial role in protecting against allergic diseases [81], highlighting the relevance of future studies on BA-mediated induction of regulatory PCs in early life and long-term experiments. Possibly, induction of IL-10 + PCs by BA in early life might be an effective way to protect against allergy and autoimmunity, especially in the long run.
In conclusion, we showed that the microbial metabolite BA induces the differentiation of regulatory IL-10 + IgM + PCs that was associated with a reduced class switch to antigen-specific pathogenic murine IgG2 antibodies. Contributing mechanisms to the induction of regulatory IL-10 + IgM + PCs likely encompass the downregulation of HDAC3 activity. Additionally, reduced mitochondrial superoxide levels after BA treatment are necessary for PC differentiation, but not IL-10 induction. Thus, epigenetic modulation of PC differentiation towards a more regulatory phenotype by BA or targeted inhibition of HDAC3 might be a potential therapeutic target in autoimmune and allergic diseases.